Method for treating diabetic ulcers

ABSTRACT

A method and means have been developed to deliver a therapeutic dose or dosages of the angiogenic molecule, Vascular Endothelial Growth Factor (VEGF) that results in a statically significant decrease in the time to achieve 100% wound closure and accelerates the rate of healing in experimental diabetic ulcers. Toxicity is evaluated by measuring any local inflammatory response at the wound site, the systemic absorption of VEGF, and the effect on distant organs that may be particularly susceptible to VEGF therapy (e.g., retinopathy and hepatitis) The angiogenic response is quantified by measuring the change in collagen deposition, epithelialization, and the closure rates of diabetic ulcers after therapeutic dosing with ADV-VEGF. Sustained administration of VEGF stimulates and accelerates the healing process as evidenced by a reduced time to complete healing (defined by 100% epithelialization and no drainage) in experimental diabetic ulcers, with minimal to no toxicity. Important features of the method and reagents for use therein are that the VEGF is released into the ulcer in a sufficient quantity over a period of time for at least two to six weeks, or to closure of the wound.

[0001] This application claims priority to U.S. Ser. No. 60/363,584filed Mar. 12, 2002.

[0002] The United States government has certain rights in this inventionby virtue of a NIH, National Institute of Diabetes and Digestive andKidney Diseases (NIDDK) grant R21DK060214-01 and NIH, National Instituteof Diabetes and Digestive and Kidney Diseases (NIDDK) grantK08DK059424-01 to Dr. Harold Brem.

BACKGROUND OF THE INVENTION

[0003] The present invention is a method of tissue engineering, andspecifically relates to administration of angiogenic factors directly todiabetic wound ulcers, in an effective amount for a sustained period oftime effective to promote closure.

[0004] Among the 16 million diabetic patients (diagnosed andundiagnosed) in the United States, an estimated 1200 amputations areperformed each week (Pecoraro, et al., Diabetes Care. 1990; 13:213-521);84% of which are preceded by a foot ulcer. Limb amputation in diabeticsis associated with an increased risk for further amputation, with afive-year mortality rate of 39 to 68% (Reiber et al. Diabetes inAmerica. Washington, D.C.; U.S. Government Printing Office,1995:409-428). The direct costs of a lower extremity amputation rangefrom $20,000 to $60,000. When failed vascular reconstruction,rehabilitation, and lost productivity within society are considered,these costs greatly exceed financial analysis. The grave consequences,pain, and suffering endured by patients with diabetic foot ulcersmandate determination of the best combination of therapies to preventprogression and, consequent occurrence of these complications.

[0005] In addition to amputation, the need to have accelerated healingin diabetic patients with foot ulcers is accentuated because thesepatients have impaired immunity (Geerlings et al. FEMS Immunol MedMicrobiol. 1999;3-4:259-265; Feige et al., EXS. 1996;77:359-373; Bessmanet al., J Diabetes Complications. 1992;4:258-262; Abraham, et al., JDermatol. 1990;7440-447; Loots et al., J Invest Dermatol.1998;5:850-857; Brown et al., J Surg Research 1994;56:562-570;Greenhalgh et al., Am J Pathol 1990;1361235-1246). Since unhealed openwounds are portals for systemic infection, they can have particularlydevastating effects for the diabetic patient.

[0006] It is well known that diabetic patients are predisposed toulceration. This predisposition has multiple etiologies, includingendothelial cell dysfunction, accelerated atherosclerosis, andperipheral neuropathy, which relate to the endothelium and contribute todeficits in healing. One of the most central etiologies for thispredisposition is the reduced angiogenic response in the diabeticpatient.

[0007] Recently, local use of growth factors has been shown to bepromising for the treatment of diabetic ulcers. Two new agents have beenFDA approved for the treatment of diabetic foot ulcers. The first,platelet derived growth factor (PDGF-BB), has shown efficacy. Thesecond, human skin equivalent, has also shown efficacy in the treatmentof diabetic foot ulcers. However, despite their successes, there remainseveral significant problems with these therapies: neither hasdemonstrated efficacy in ischemic diabetic foot ulcers, and both have aminimum failure rate of 45% in well vascularized limbs. Although this isbetter than the failure rate of standard therapies (i.e., off-loadingand saline dressing), the number of amputations and non-healed diabeticfoot ulcers remains excessive. Although both of these therapies havealso demonstrated that local therapy is clinically effective in thetreatment of diabetic foot ulcers, more therapies are clearly needed.

[0008] Therefore, it is an object of the present invention to provide amethod and means to provide increased blood flow to diabetic ulcers, andthereby promote wound closure.

SUMMARY OF THE INVENTION

[0009] A method and means have been developed to deliver a therapeuticdose or dosages of the angiogenic molecule, Vascular Endothelial GrowthFactor (VEGF) that results in a statistically significant decrease inthe time to achieve substantially 100% wound closure and accelerates therate of healing in experimental diabetic ulcers. Toxicity is evaluatedby measuring any local inflammatory response at the wound site, thesystemic absorption of VEGF, and the effect on distant organs that maybe particularly susceptible to VEGF therapy (e.g., retinopathy andhepatitis) The angiogenic response is quantified by measuring the changein collagen deposition, epithelialization, and the closure rates ofdiabetic ulcers after therapeutic dosing with adenoviral vector(ADV)-VEGF or VEGF. Sustained administration of VEGF stimulates andaccelerates the healing process as evidenced by a reduced time tocomplete healing (defined by 100% epithelialization and no drainage) inexperimental diabetic ulcers, with minimal to no toxicity. Importantfeatures of the method and reagents for use therein are that the VEGF isreleased into the ulcer in a sufficient quantity over a period of timefor at least two to six weeks, or to closure of the wound. The VEGF canbe administered directly or as the gene which is expressed at the sitein need of treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010]FIG. 1 is a graph of the proliferation of human vascularendothelial cells (HUVECs) stimulated with the supernatant of JC cellsinfected with adenoviral vectors expressing VEGF, as a function ofnumber of cells (absorption) versus dilution of conditioned supernatant,compared to control (beta-galactosidase).

DETAILED DESCRIPTION OF THE INVENTION

[0011] VEGF and Angiogenesis

[0012] Local angiogenic therapy is used to treat one of the primaryetiologies of complications in patients with diabetic foot ulcers,decreased angiogenesis in the wound. It has been well established thatdistal bypass alone is not enough to sufficiently accelerate the healingof most diabetic foot ulcers.

[0013] There are multiple physiological processes that result indecreased angiogenesis in a diabetic ulcer; including:

[0014] 1. Decreased VEGF expression in experimental diabetic wounds.VEGF normally stimulates endothelial cell proliferation and experimentaldiabetic wounds show a marked decrease in angiogenesis (Frank et al., JBiol Chem 1995;270:12607-12613; Tsuboi, et al., J Explorer Med1990;172:245-251).

[0015] 2. Endothelial dysfunction results in several metabolic anomaliesin diabetes, including oxidative stress, hyperglycemic pseudohypoxia,nonenzymatic glycosylation, and activation of the coagulation cascade(Stehouwer et al., Cardiovasc Res 1997;34:55-68)

[0016] 3. Accelerated atherosclerosis may impair function by damagingthe transduction cascades that synthesize nitrous oxide or by decreasingexpression of nitrous oxide synthase, consistent with the finding ofdecreased nitrous oxide synthase expression in skin endothelium fromdiabetic patients (Liao, et al., J Biol Chem 1995;270:319-324; Veves etal., Diabetes 1998;47:457-463).

[0017] 4. Neuropathy is associated with endothelium dependent andindependent dysfunction in diabetic patients, predisposing them to footulceration (Pasini et al., Angiology 1996;47:569-577).

[0018] 5. Arterial occlusive disease is associated with peripheralneuropathy, manifested as slower conduction velocity of motor andsensory nerves and depression of automatic responses.

[0019] Other factors are thought to be significant variables in thetreatment of diabetic wound ulcers, especially the high pressure withinthe wound, which contributes to the ischemia and neuropathy, although ithas not been apparent what single factor, if any, could be effective inpromoting wound closure.

[0020] VEGF has been shown to be efficacious in the treatment of cardiacischemia and leg ischemia, and it has demonstrated effectiveness in thetreatment of diabetic limbs. However, it has not previously been testedin diabetic ulcers. VEGF possesses certain key advantages for use indiabetic foot ulcers over other angiogenic agents, for example, basicfibroblast growth factor (bFGF). Although bFGF has been one of the mostwidely studied angiogenic growth factors for the treatment ofexperimental diabetic ulcers (Albertson et al. Surgery 1993;114:368-373), other chronic wounds (Quirinia, et al., Ann Surg1998;227:446-454), and accelerating acute wound healing (Hebda et al., JInvest Dermatol 1990;95:626-631), it has not been shown to be effectivein treating human diabetic ulcers (Richard, et al., Diabetes Care1995;18:64-69). Unlike VEGF, bFGF is not acid stable, which is criticalin the low pH environment of a diabetic wound (Ferrara, et al., BiochemBiophys Res Comm 1989; 161:851 -858). The impaired wound healing indiabetics may be due to fibroblast dysfunction (Lerman et al. Am. J.Pathol. 2003; 162:303-312). Fibroblasts from diabetic db/db micemaintain selective impairments in multiple cellular processes which areaccentuated by hypoxic environments such as those in a healing wound.These impairments include a severe reduction in VEGF expression, andrelease in response to injury. These changes were only observed afterthe development of the diabetic phenotype in these animals.Additionally, VEGF has been shown to be a more potent angiogenicmolecule than bFGF in experimental diabetic wound (Corral, et al., ArchSurg. 199; 134:200-5). Also, VEGF levels are apparently decreased byendogenous proteases (Lauer, et al., J Invest Derm 2000;115:12-18) inchronic wounds. This provides a basis from which to develop gene therapywith VEGF for use in diabetic wounds. The intense and sustained localsynthesis of VEGF by local angiogenic therapy should obviate theproteolytic degradation of endogenous VEGF in the diabetic wound.

[0021] The role of VEGF in angiogenesis reflects its function as anendothelial cell mitogen, (Ferrara N., J Mol Med 1999;77:524-543;Gospodarowicz D, et al., Proc Natl Acad Sci 1989;86:7311-7315)chemotactic agent, (Leung D W, et al., Science 1989;246: 1306-1309; KeckP J, et al., Science 1989;246: 1309-1312) and inducer of vascularpermeability (Connolly D T, et al., J Clin Invest 1989;84: 1470-1478;Yoshida A, et al., Growth Factors 1996;13:57-64; Senger D R, et al.,Cancer Res 1990;50:1774-1778). It exists in many isoforms with a commonamino terminus that contains a signal sequence that allows the proteinto be secreted. VEGF 165 is the most common isoform and is preferred,although it is understood that other equivalent forms of VEGF are knownand could be used alone or in combination with each other as describedherein.

[0022] Many of the cells (Banks, et al., Br J Cancer 1998;77:956-964,Nathan, J Clin Invest 1987;79:319-326; Berse, et al., Mol Biol Cell1992;3:211 -220; Leibovich, et al., Am J Pathol 1975;78:71-100; Koch, etal., Science 1993;258:1798-1801; Uchida, et al., Am J Physiol1994;266:F81-F88; Namiki, et al., J Biol Chem 1995;270:31189-31195;Nissen, et al., Am J Path 1998;152:1445-1452; Brogi, et al., Circulation1994;90:649-652; Stavri, et al. Circulation 1995;92:11-14) recruitedinto a wound synthesize VEGF. VEGF serves distinct paracrine andautocrine roles upon endothelial cells. By stimulating the endothelialcells, multiple phases of the angiogenic cascade are enhanced by VEGF.VEGF has been shown in experimental studies to enhance vascularizationof both autologous bone grafts and skin flaps in rats ( Padubidri, etal., Ann Plast Surg 1996;37:604-611; Li, et al. Surg Forum.1999;50:586-587). One of the mediators of VEGF activity, nitric oxide,enhances collagen deposition in diabetic wounds, (Witte, et al., SurgForum 1997:48:665-667) and may restore endothelial function to improveboth nerve conduction and tissue oxygenation. This supports the conceptthat VEGF enhances wound healing primarily by stimulating angiogenesisand possible secondary stimulation of collagen production.

[0023] Local Sustained Release of VEGF in a Diabetic Wound

[0024] In the twelve years that VEGF has been available, local therapywith recombinant forms has not yet been shown to be effective for woundclosure. One limitation to this technique is that it fails to sustainsufficient levels of VEGF in the wound for a significant period of time.Gene therapy is an effective means for delivering VEGF in a sustainedrelease fashion in vivo. ADV-VEGF has already been tested in patientswith cardiac ischemia and leg ischemia to stimulate angiogenesis.Additionally, it has also been tested for diabetic ischemia andneuropathy. Other means of obtaining sustained release of an effectiveamount of compound include providing sustained release formulations suchas polymeric delivery systems, mini-pumps, and hydrogels. These can beloaded with VEGF, injected or implanted into the ulcers, where the VEGFis released over a therapeutically effective time period.

[0025] The principle of gene therapy is that a therapeutic gene mustfirst be efficiently delivered to the specific target cell (Nabel, etal., Science. 1990;249:1285-1288). Second, it must be expressed andsustained at a certain level to achieve its therapeutic purpose (Sauter,et al., Proc Natl Acad Sci USA. 2000;9:4802-4807).

[0026] The principle components of gene therapy are a vector or othermeans of delivering a nucleic acid of interest, and the nucleic acid.Many appropriate viral vectors are known, most of which are adenoviralvectors, adeno-associated viral vectors or retroviral vectors. Othermeans of delivery include liposomes, direct delivery of naked DNA, andhydrogels. The vectors will typically include a promoter that cancontain enhancers, inverted terminal repeats (ITRs), induciblepromoters, and polyA sequences, followed by a termination sequence. Allof these are known to those skilled in the art, and commerciallyavailable or described in the literature.

[0027] The discovery that naked DNA is taken up by muscle cells andtransiently expressed in vivo, was reported twelve years ago, by Wolff,al, et, Science, 1990;247,1465-1468,; and Wolff, Nature, 1991;352,815-818.

[0028] Plasmid DNA, which can function episomally, has been used withliposome encapsulation, CaPO₄ precipitation, and electroporation as analternative to viral transfections. Clinical trials with liposomeencapsulated DNA in treating melanoma illustrates this approach to genetherapy, as reported by Nabel, et al., Proc. Nat. Acad. Sci. USA.,1993;90, 11307-11311 and Wolff, Experimental Neurology, 1992;115,400-413, also reported expression of plasmid DNA. There have been manyconfirmatory reports since the initial studies.

[0029] Viral vectors are preferred for gene therapy. Human adenoviruseshave a 36-kilobase double-stranded DNA genome that undergoes a highlyregulated program of gene expression during the normal life cycle of thevirus. The advantages of adenoviruses over other chemical, physical, orbiological gene transfer techniques include several unique features ofthis system (Molnar-Kimber, et al., Hum Gene Ther Sep. 20,1998;9(14):2121-33). First, adenoviruses infect human skin cells at morethan 95% efficiency and do not require that cells are dividing, making alengthy selection process unnecessary (Kozarsky, et al., Curr Opin GenetDev June 1993;3(3):499-503; Mulligan, Science May 14,1993;260(5110):926-32; Kremer, Gene Ther 1995;2:564-5; Yang, et al.,Immunity August 1994;1(5):433-42; Mitani, et al., Proc Natl Acad Sci USAApr 25, 1995;92(9):3854-8). Second, adenoviruses remain episomal andthus do not normally integrate into the human genome (Bett, et al., JVirol October 1993;67(10):5911-21 Losordo, et al., Am Heart J.1999;138:132-141). Third, adenovirus-mediated gene expression inkeratinocytes, melanocytes, and fibroblasts remains stable in vitro forat least 2-6 weeks, depending on the proliferation rate of cells (Feng,et al., Cancer Res May 15, 1995;55(10):2024-8). Adenoviral vectors arecommonly constructed by deletion of the essential ELAM-1 gene to preventviral replication.

[0030] For the purpose of wound healing, ADV-VEGF gene therapy may offersubstantial advantages, one of which is its ability to transduce bothresting and dividing cells at very high efficiency without integrationinto the host cell's genome. The inflammatory response that is oftenassociated with adenoviral mediated gene transfer for the induction ofendogenous growth factor overexpression may actually enhance woundhealing, since wound healing is itself fundamentally an inflammatoryresponse. Furthermore, the limited duration of high level transgeneexpression of a growth factor known to have significant vulneraryeffects (like VEGF) may be all that is necessary to affect woundhealing. Recombinant ADV can be generated in high titers, is known toinfect both resting and dividing cells with high efficiency, and itstransgene expression is limited by the host's cellular immunity. Thehigh prevalence of anti-adenovirus antibodies in the general populationwas thought to preclude adenovirus-mediated gene therapy. However, itwas shown that local injection of rADV in pre-sensitized hosts resultsin levels of transgene expression at the injection site comparable tothat of native animals (Bramson, et al., Gene Ther 1997;4:1069-76).These findings stress both the applicability of this local gene and thepotential for repeated treatments, if required. Although, for effectivegene therapy, the transgene may need to be expressed in a higherpercentage of cells in the target population, lower rates of expressionmay be sufficient if the gene product exerts a paracrine effect onneighboring cells. Toxicity to tissues other than the target, due to thebroad tropism of the adenovirus, is usually seen only with systemicadministration.

[0031] Adenoviruses can transduce both dividing and nondividing cellsand the viral genome, remains episomal and does not integrate into hostchromosomes. They can infect a broad range of human cells such as thosein skin, lung, liver, brain, and blood vessels. It has recently beenshown that adenoviral vectors are very effective for in vivo genetransfer to porcine and other experimental skin wounds and nearlymicrogram quantities of therapeutic protein can be expressed in thewound microenvironment (Liechty, et al., Wound Rep Reg 1999;7:148-53;Liechty, et al., J Invest Dermatol 1999;113:375-83).

[0032] The usefulness of administration of ADV-VEGF in reducing the timeneeded for wound healing, as described in U.S. Ser. No. 60/363,584 filedMar. 12, 2002, was recently confirmed in experimentally-induced excisionwounds in diabetic mice (Romano Di Peppe, et al Gene Therapy, 9:1271-1277 (October 2002)). A statistically significant reduction inwound healing time as compared to untreated mice was noted as early as 3days after treatment, in mice treated with 10⁸ p.f.u. of AdCMV.VEGF₁₆₅directly on the wound. There was also an increase in VEGF expression inthe wounded skin and significant angiogenic response. The concentrationof AD-VEGF can be titrated accordingly to result in expression of aneffective amount of VEGF.

[0033] Polymeric Matrices

[0034] Alternatively, the VEGF is delivered using a sustained releasedevice. Both non-biodegradable and biodegradable matrices can be usedfor delivery of genes, although biodegradable matrices are preferred.These may be natural or synthetic polymers, although synthetic polymersare preferred due to the better characterization of degradation andrelease profiles. The polymer is selected based on the period over whichrelease is desired, generally in the range of at least two to six weeks,although longer periods may be desirable. In some cases linear releasemay be most useful, although in others a pulse release or “bulk release”may provided more effective results. The polymer may be in the form of ahydrogel (typically in absorbing up to about 90% by weight of water),and can optionally be crosslinked with multivalent ions or polymers.

[0035] High molecular weight genes can be delivered partially bydiffusion but mainly by degradation of the polymeric system. In thiscase, biodegradable polymers, bioerodible hydrogels, and proteindelivery systems are particularly preferred. Examples ofnon-biodegradable polymers include ethylene vinyl acetate,poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof.Examples of biodegradable polymers include synthetic polymers such ashydroxyacid polymers, for example, polymers of lactic acid and glycolicacid, polyanhydrides, poly(ortho)esters, polyurethanes, poly(buticacid), poly(valeric acid), and poly(lactide-co-caprolactone), andnatural polymers such as alginate and other polysaccharides includingdextran and cellulose, collagen, chemical derivatives thereof(substitutions, additions of chemical groups, for example, alkyl,alkylene, hydroxylations, oxidations, and other modifications routinelymade by those skilled in the art), albumin and other hydrophilicproteins, zein and other prolamines and hydrophobic proteins, copolymersand mixtures thereof. In general, these materials degrade either byenzymatic hydrolysis or exposure to water in vivo, by surface or bulkerosion.

[0036] In the preferred embodiment, the polymeric matrix is amicroparticle between nanometers and one millimeter in diameter, morepreferably between 0.5 and 100 microns for administration via injection.The microparticles can be microspheres, where the gene is dispersedwithin a solid polymeric matrix, or microcapsules, where the core is ofa different material than the polymeric shell, and the gene is dispersedor suspended in the core, which may be liquid or solid in nature. Unlessspecifically defined herein, microparticles, microspheres, andmicrocapsules are used interchangeably.

[0037] Alternatively, the polymer may be cast as a thin slab or film,ranging from nanometers to four centimeters, a powder produced bygrinding or other standard techniques, or even a gel such as a hydrogel.The polymer can also be in the form of a coating or part of a stent orcatheter, vascular graft, or other prosthetic device.

[0038] The matrices can be formed by solvent evaporation, spray drying,solvent extraction and other methods known to those skilled in the art.

[0039] The release of VEGF from fibrin-based biomaterials wasdemonstrated by Wong et al., Thromb Haemost 89(3):573-82 March 2003).Fibrin-based biomaterial preparations can be used as provisional growthmatrices for cells important in tissue repair during wound healing invivo. VEGF was incorporated into the fibrin biomaterials prior toformation of the Fibrin Sealant clots. Clotting resulted in sustainedrelease of VEGF causing angiogenic activity.

[0040] Prolonged controlled release has been achieved using severaldifferent devices. Examples include mini-implantable pumps for a varietyof drugs especially chemotherapeutics and highly potent neuroactivedrugs, silicon tubing with release controlling pores in the ends forbirth control agents, and co-axial implants. Currently approved infusionprocedures generally use an externally-worn or implanted pump. DUROS®sufentanil, an osmotic pump designed for 100-day delivery of sufentanil,is currently undergoing clinical testing. This implant is much smallerand easier to administer, and is described in WO 00/54745.

[0041] Effective Dosages of VEGF

[0042] Unlike in the case of an ischemic ulcer, for a diabetic ulcer,the challenge remains to keep a steady rate of angiogenic stimulationwith minimal toxicity. At this stage in the field of wound healing, itappears that diabetic wounds have impaired angiogenesis. VEGFadministered locally into the wound is the safest and most effectivemethod to stimulate angiogenesis and to facilitate healing the diabeticwound.

[0043] The use of VEGF₁₆₅ has been established in multiple models ofangiogenesis (Kaner, et al., Am J Respir Cell Mol Biol. 2000;6:640-641;Muhlhauser, et al., Circ Res. 1995;6:1077-1086) Human VEGF₁₆₅ molecule(rather than the murine molecule) has already been established in thepreclinical studies for cardiac ischemia (using a porcine model, Esakol,et al., Hum Gene Ther. 1999;10:2307-2314; Lopez, et al., Cardiovasc Res.1998;40:272-281; Baumgartner, et al., Annu Rev Physiol. 2001;63:427-450;Losordo, et al., Am Heart J. 1999;138:132-141; Pearlman, et al., NatMed. 1995;1:1085-1089; Hariawala, et al., J Surg Res. 1996;63:77-82),and a dog model (Banai, et al., Circulation. 1994;89:2183-2189;Lazarous, et al., Cardiovasc Res. 1999:44;294-302) which led to itseventual use and demonstrated efficacy in humans (Esakof, et al., HumGene Ther. 1999;10:2307-2314; Sumes, et al., Ann Thorac Surg1999;68:830-7; Losordo, et al., Circulation. 1998;98:2800-2804;Baumgartner, et al., Am Heart J. 1999;138:132-41; Hendel, et al.,Circulation. 2000;101:118-121; Hammond, et al., Cardiovasc Res.2001;49:561-567; Sylven, et al., Coron Artery Dis. 2001;12:239-243; Tio,et al., Human Gene Therapy. 1999;10:2953-2960; Laitinen, et al., HumGene Ther. 2000;11:263-270; Vale, et al., Circulation.2000;102:965-974).

[0044] Similarly, VEGF₁₆₅ was used in preclinical studies for limbischemia and atherosclerosis in rabbits (Gennaro et al., CirculationJan. 21, 2003;107(2):230-3; Takeshita, et al., Biochem Biophys ResCommun. 1996;227:628-635; Tsurumi, et al., Circulation.1996;94:3281-3290; Takeshita, et al., J Clin Invest. 1994;93:662-670;Banters, et al., Circulation. 1995;91:2802-2809; Bauters, et al., J VaseSurg. 1995;21:314-325; Takeshita, et al., Circulation.1994;90(suppl1II):II-228-234; Vincent, Circulation. 2000;102:2255-2261)and diabetic mice, which led to its eventual clinical trial testing anddemonstrated efficacy for patients (Simovic, et al., Arch Neurol.2001;58:761-768; Isner, et al., J Vasc Surg. 1998;28:964-75). Recently,human VEGF has been used in diabetes (e.g., for diabetic neuropathy inrats, (Samii, et al., Neurosci Lett. 1999;262:159-162; Schratzberger, etal., J Clin Invest. 2001;107:1083-1092) for diabetes-related impairmentof angiogenesis in mice, (Rivard, et al., Am J Pathol. 1999;154:355-363)for ischemic neuropathy in rabbits, (Schratzberger, et al., Nat Med.2000;4:405-413) and in patients (Baumgartner, et al., Circulation.1998;97:1114-1123; Isner, et al., Hum Gene Ther. 1996;7:959-988; Isner,et al., Lancet. 1996;10:370-374; Schratzberger, et al., J Clin Invest.2001;107:1083-1092). Furthermore, successful treatment of diabeticneuropathy in rodents led to human trials for efficacy of chronicischemic neuropathy in patients (Simovic, et al., Arch Neurol.2001;58:761-768). A vast amount of literature has demonstrated that theuse of human VEGF in a mouse shows functionality due to a high degree ofhomology between the two species-specific proteins (90%, data fromBLAST® protein homology searching). Finally, according to the EuropeanMolecular Biology Laboratory (EMBL) database, the amino acid of humanVEGF protein and murine VEGF proteins demonstrate only one amino aciddifference (215 vs. 214).

[0045] An effective dosage can be determined by extrapolation based onanimal studies, for example, using a mouse model. In previous localtherapy studies, the use of full thickness excisional wounds in diabeticmice has been shown to be useful for developing therapies for eventualuse in patients. The mouse model has been successful in bringing growthfactor therapy from the lab to the bedside. Both FDA approved drugs thatare currently efficacious for diabetic ulcers (PDGF-BB and human skinequivalent) utilized the murine model in the vast majority of theirpreclinical testing.

[0046] The C57BL/KsJ db/db mouse is a particularly useful model since ithas been shown to have decreased angiogenesis (Altavilla, et al.,Diabetes. 2001;50:667-673; Coleman, Diabetes. 1982;31 (Suppl: 1 Pt2):1-6). Tissue repair in C57BL/KsJ db/db mice has proven to be aclinically relevant model of impaired wound healing. The animals exhibitseveral characteristics of adult onset diabetes, including obesity,insulin-resistant hyperglycemia and markedly delayed wound closure.

[0047] C57BL/KsJ-db/db mice, homozygous for the diabetes spontaneousmutation, become identifiably obese around 3 to 4 weeks of age.Elevations of plasma insulin begin at 10 to 14 days and of blood sugarat 4 to 8 weeks. Homozygous mutant mice are polyphagic, polydipsic, andpolyuric. The course of the disease is markedly influenced by geneticbackground. A number of features are observed on the C57BL/KsJ db/dbbackground, including an uncontrolled rise in blood sugar, severedepletion of the insulin-producing beta-cells of the pancreatic islets,and death by 10 months of age. Exogenous insulin fails to control bloodglucose levels and gluconeogenic enzyme activity increases. The diabeticmutation is a result of a point mutation in the leptin receptor gene,lepr. This point mutation promotes abnormal splicing creating a stopcodon that shortens the intracellular domain of the receptor, so thatits signaling capacity is curtailed. The ligand, Leptin, has been shownto be a key weight control hormone that takes a mutant form in the mouseobesity mutation, Lepob (JAX Mice database:http://jaxmice.jax.org/jaxmice-cgi/jaxmicedb.cgi).

[0048] C57BL/KsJ-db/db mice exhibit characteristics similar to those ofhuman adult onset diabetes (NIDDM Type II) as a result of a singleautosomal recessive mutation on chromosome 4. Only the homozygousanimals develop diabetes. This strain also expresses lower levels ofseveral growth factors and receptors, accounting, at least in part, forthe reduced rate of healing (Werner, et al., J Invest Dermatol1994;103:469-473).

[0049] The streptozotocin diabetic mouse is another model for studyingthe pathology of diabetes. Mice are rendered diabetic by intraperitonealinjection of streptozotocin administered for five consecutive days.Streptozotocin-treated mice become hyperglycemic and also show impairedwound healing when compared to healthy animals (Matsuda et al. J Exp Med1998; 187:297-306; Brown et al Am J Pathol 1997; 151:715-724). Thestreptozotocin-induced diabetic mouse has been widely studied and isknown to those of skill in the art.

[0050] The diabetic mouse model (Geerlings et al., FEMS Immunol MedMicrobial. 1999;3-4:259-265; Feige, et al., EXS. 1996;77:359-373;Bessman, J Diabetes Complications. 1992;4:258-262; Abraham, et al., JDermatol. 1990;7440-447; Loots, et al., J Invest Dermatol.1998;5:850-857; Brown, et al., J Surg Research 1994;56:562-570;Greenhalgh, et al., Am J Pathol 1990;136:1235-1246; Tsuboi, et al., JExplorer Med 1990;172:245-251; Matuxzewska, et al., Pharm Res1994;11:65-71; Darby, et al., Int J Biochem Cell Biol 1997;29:191-200;Livant, et al., J Clin Invest. 2000;105:1537-1545; Yamamota, et al.,Europ J Pharm 1996;302:53-60; Wetzler, et al., J Invest Dermatol.2000;115:245-253; Sun, et al., J Invest Dermatol 1997; 108:313-318; Sun,et al., J Invest Dermatol. 1996;106:232-237; Zykova, et al., Diabetes.2000;49:1461-1458; Beer, et al., J Invest Dermatol. 1997;109:132-138)has been widely accepted in the study of therapeutic agents that may beeffective in the treatment of chronic wounds, it has been successfullyused in preclinical testing for other growth factor therapies, and itoffers a good model for patients with diabetic foot ulcers.

[0051] The present invention will be further understood by reference tothe following non-limiting examples.

EXAMPLE 1 Non Diabetic Wounds are Angiogenic Dependent and have DelayedContraction with Angiogenic Inhibitors

[0052] There are multiple stimuli for angiogenesis in the first fewhours after a wound is created, but it is usually 3 days before newvessel formation can be visualized histologically in the wound.Throughout the wound healing process, angiogenesis maintains a criticalrole in the healing response (Brem, et al., Bone Formation and RepairAmerican Academy of Orthopedic Surgeons/Rosemont, Ill., 1994. pp213-222; Arbiser. J Am Acad Dermatol 1996;34:486-497; Pettet G, et al.,Proc R. Soc Lond 1996;263: 1487-1493; Arnold, et al., Pharmacol Ther1991;52:407-422; Seifert, et al., Explorer Mol Pathol 1997;64:31-40;Folkman, Growth Factors: From Genes to Clinical Application. KarolinskaInstitute Nobel Conference Series. New York, N.Y., Raven Press, 1990, pp201-216; Folkman, et al., Inflammation: Basic Principles and ClinicalCorrelates, ed 2. New York, N.Y., Raven Press, 1992, pp 821-839; Hunt,et al., Surgery 1984;96:48-54).

[0053] Angiogenesis occurs in a time-dependent manner in relation to thewound healing process. For example, when a potent angiogenic inhibitor(e.g., TNP-470, also known as AGM-1470) is given systemically, woundclosure and breaking strength are significantly decreased in atime-dependent manner. If the angiogenesis inhibitor is given before or5 days after wound creation, there is no delay in wound closure, but ifit is given in the first 5 days after wounding a sharp delay in closureis noted.

[0054] These experiments demonstrate the importance of the initiation ofangiogenesis in contributing to the multiple processes in normal woundhealing, such as wound closure and breaking strength. In the healing ofacute wounds, these results show that the angiogenic response istime-dependent. This experiment establishes that decreased angiogenesisdelays wound closure. In chronic wounds, such as diabetic ulcers, theangiogenic response is impaired as long as the wound is present,secondary to multiple etiologies.

EXAMPLE 2 The Viral Vector

[0055] The human cDNA for VEGF was cloned into a recombinant adenovirusvector. Human umbilical vein endothelial cells (HUVEC) were harvestedfrom fresh umbilical cords using a 0.2% collagenase solution in HanksBalanced Salt Solution (HBSS) for 20 minutes at room temperature. Thecells were washed with HBSS and plated on collagen-coated (1% in PBS)tissue culture dishes in M13 medium supplemented with 20% FBS and 1mg/ml of bFGF. HUVEC from a confluent 10-cm plate were homogenized andtotal RNA was extracted with the RNEASY™ kit from QIAGEN® Inc. Firststrand of cDNA was amplified from the RNA by RT-PCR with oligo-dTprimers using the SUPERSCRIPT™ II RT PCR kit from LIFE TECHNOLOGIES®.The full-length human VEGF cDNA was then amplified by PCR withappropriate primers (sense 5′-CCCAAGCTTGCCGCCGCCATGAACTTTC TGCTGTCT-3′(SEQ ID NO:1); Hind III linker antisense,5′-GCTCTAGAATCTGGTTCCCGAAACCCTGA-3′ (SEQ ID NO:2), Xba I linker) usingpfu (plaque forming units), DNA polymerase (STRATAGENE®). The productcorresponding to the size of VEGF₁₆₅ was gel-purified and cloned intopBluescript (STRATAGENE®), to be spliced between the Hind III and Xba Isites. After confirmation by automatic sequencing, the hVEGF₁₆₅ cDNA wascloned into the ADV shuttle vector pADV RSV-bPA downstream of the RSVLTR. The ADV shuttle vector was then co-transfected with the adenovirustype 5 genome containing backbone vector pBHG10 into 293 cells (E1positive packaging cell line) by standard calcium phosphateprecipitation. The ensuing viral plaques were isolated and in vitroscreened for transgene expression and functional activity of thetransgene. A positive plaque (#11-1) was then selected and expanded. Thevirus (ADV.hVEGF₁₆₅) was released from the producer cells by threefreeze/thaw cycles and purified over a CsCl gradient. The viralparticles were measured by spectrophotometric analysis (OD₂₆₀) and theplaque-forming units (pfu) were determined by standard agarose overlayplaque assay on 293 cells.

[0056] The vector, as constructed, resulted in gene transfer,ascertaining transgene expression in target cells in culture. JC cells,a VEGF-negative murine breast cancer cell line which is easilytransducible by adenovirus, were infected with virus of differentplaques of ADV.hVEGF₁₆₅ or ADV.beta Gal (negative control) with an MOIof 100. VEGF was measured after 48 hours in the conditioned supernatantby hVEGF ELISA (R&D Systems), according to the manufacturer'sinstructions. The ELISA results for hVEGF of conditioned supernatant ofJC cells transduced are:

[0057] 1) Virus of different plaques of ADV.hVEGF₁₆₅: (plaques 11-1;11-2; 11-3; 11-9; 13-1; 16-1);

[0058] 2) Control vector (beta-Gal); and supernatant of uninfected JCcells. (VEGF standard curve with recombinant VEGF protein supplied bythe manufacturer).

[0059] The transgene for VEGF stimulates endothelial cell proliferationin vitro. The function of the transgene was assessed through inductionof HUVEC proliferation by conditioned supernatant of ADV.hVEGF₁₆₅ orADV.beta Gal transduced JC cells, respectively. HUVECs were seeded inflat-bottom well plates pre-coated with 1% gelatin (in PBS) at 2×10³cells/well and serum-starved overnight (M13 medium without FBS).Conditioned supernatant was then added in several dilutions and cellswere cultured for an additional 48 hours in M13 supplemented with 20%FBS, but no growth factors. HUVEC proliferation was measured by atetrazolium-based assay at an absorption of 450 nm according to themanufacturer's instructions, EZ4U™ kit by Biomedica. Measurements ofHUVEC proliferation by a tetrazolium-based assay after a 48-hourstimulation with log 2 dilution of conditioned supernatant of JC cellswere transduced with:

[0060] 1) virus of different plaques of ADV.hVEGF₁₆₅: (plaques 11-1,13-1, 16-1); and

[0061] 2) control vector (b-Gal).

[0062] Addition of recombinant VEGF protein (Pepro Tech Inc.) to theculture medium in a log 2 dilution series from 100 ng/ml to <1 ng/ml wasused as a positive control.

[0063] The results are shown in FIG. 1.

EXAMPLE 3 ADV-VEGF Leads to Angiogenesis

[0064] Mice received subcutaneous injections of ADV-VEGF in one thighand ADV.LacZ in the other. At various time intervals, these mice weresacrificed and perflusion-fixed. The thighs were harvested and processedfor histology and immunohistochemistry. Histology revealed nosignificant changes in the control (ADV.LacZ)P group and marked presenceof new blood vessels in the ADV-VEGF group.

[0065] Thus, it was established that ADV-VEGF infected cells secretedhuman VEGF₁₆₅ and that this protein product stimulated endothelial cellproliferation in vitro and angiogenesis in vivo, without local necrosis.

[0066] Examples 4-7 use a mouse model. For all mouse wound experiments,mice were purchased from Jackson Laboratories, and housed one per cage.Animals were anesthetized in a chamber in preparation for allexperiments and shaved the day before experimentation. Full thicknesswounds were made with a template of 0.8 or 1.4 cm in diameter. Allwounds were created in exactly the same location, 15 mm from the lastcervical vertebra on the mouse dorsum. The marked area was excised withscissors to include the epidermis, dermis, and panniculus carnosus. Thewounds were then photographed on the day of wounding. The photographsand digital images were taken with a NIKON® 35 mm camera and a NIKON®digital camera, respectively, at a fixed distance from the wound. Aphoto ruler was placed in the field of the photograph and labeled withthe mouse's identification number and date. Digital photos were used toassess length and width measurements and the area was calculated fromplanimetry. Length and width were also recorded in the lab notebook atthe time of photography. The data included in these studies aremaintained in a database, created with MICROSOFT ACCESS®, on a DELL™Dimension XPS R450 Computer, and backed up on an IOMEGA® Jaz Drive. Bothfilm and digital photographs of each mouse were printed within 7 days ofbeing taken and kept in a secure area. The digital images were measuredusing Med-Data Systems' Wound Imager program. An identical data systemis utilized to follow clinical patients treated for diabetic footulcers. In all experiments, the ADV-VEGF was injected into 4 quadrantsof the dermis of the newly created wound edge utilizing a Hamilton™microliter syringe in 4 equal volumes.

[0067] This model is analogous to clinical situations where patients areroutinely treated for diabetic foot ulcers, where the wound is debridedprior to initiation of growth factor therapy (PDGF-BAL) or cell therapy(human skin equivalent comprising cultured keratinocytes and fibroblastson type I collagen). The emphasis is on converting a chronic wound orulcer into an acute wound, in part, by debridement of tissue, leavingfresh wound edges. This approach is a standard of care and is the basisof the experimental design, allowing for the maximally effectiveinjection of VEGF into the sharply excised wound edges.

EXAMPLE 4 Effect of ADV-VEGF

[0068] The dosage of 5×10¹¹ viral particles (VPs) of ADV-VEGF was usedbecause this was the maximal amount of viral particles that could beplaced in a limited volume (up to 400 μl, which is the maximum that canbe administered without the volume affecting the wound). This dosage wasalso based on multiple other preclinical and clinical trials performedat our institution, which determined this to be safe for ADV particles.It has become clear that the total load of ADV particles contributes totoxicity. Therefore, the FDA has requested that experiments use viralparticles rather than pfus. Pfu is a biological assay measuring activityof the virus, which can vary by up to one log using the same batch ofvirus in different assays. If different batches of virus are used, theremay be a significant variation in particle measurements. To prevent thispotential problem from occurring and to keep the loads constant, thesame batch of virus was used for all experiments.

[0069] Six groups, comprising 5 mice each, were used. Wounds of 0.8 cmwere made with a template, as described above. All, except group F, usedthe C57BL/KsJ-db/db mouse model.

[0070] Group A: (ADV-VEGF) received 5×10¹¹ ADV-VEGF VPs at the time ofwounding. The wounds closed on average in 13.6±days.

[0071] Group B: (ADV-DL312, control null vector) received 5×10¹¹ VPs atthe time of wounding. The wounds closed in 26.9±0.4 days.

[0072] Group C: (saline control) received PBS and their wounds closed in26 days, on average.

[0073] Group D: (2 ^(nd) injection of ADV-VEGF) received a secondinjection of ADV-VEGF 7 days after wounding. The results were notstatistically different from Group A.

[0074] Group E: (human recombinant VEGF) received 2.5 μg recombinantVEGF at the time of wounding. The wounds closed at a rate similar toGroups B and C.

[0075] Group F: (wild type, non-diabetic mice) received saline. Thewounds closed in 12 days, on average.

[0076] The results show that treatment with ADV-VEGF resulted in a(highly) significant reduction in time to closure of wounds in diabeticmice (p<0.01, Student's t-test and nonparametric Mann-Whitney/Wilcoxon'stest). Although these results suggested that a single treatment withADV-VEGF was maximally effective in accelerating wound closure, thestudy was limited.

EXAMPLE 5 Dose-Response Study

[0077] Despite the limitations of the above experiment and the smallnumber of mice, the promising results motivated determining if a lowerdose may be effective. In order to determine if gene therapy iseffective, it is first necessary to perform a dose-response experiment.This allows one to establish that there is a causal relationship betweenthe therapy and its therapeutic effects. It was found that in thesesmall 0.8 cm wounds 5×10¹⁰ and 5×10¹¹ VPs of ADV-VEGF both significantlyaccelerated the closure of diabetic ulcers in db/db male mice, ascompared to 1.6×10¹⁰ ADV-VEGF VPs and PBS.

[0078] There was a significantly (p<0.05) greater improvement in therate of healing for the high dose group, compared to the control group.

[0079] A limitation in this experimental design was that the occlusivedressing placed on the wound after each photograph was taken resulted inbroad contamination with culture-proven gross purulence with Pseudomonasaeruginosa. The technique was modified to utilize only a clear occlusivedressing during the first 5 days after the wound was made. No infectionhas been seen in subsequent experiments. However, this provided aserendipitous and important finding. This experiment suggests thatADV-VEGF may reverse bacterial contamination of a wound. This isparticularly important, because almost all patients with diabetic footulcers have some degree of infection or, more specifically, bacterialcontamination, which is the single greatest contributing factor to themorbidity and mortality related to these wounds.

EXAMPLE 6 Effect of VEGF on Larger Wounds

[0080] Example 5 established that even a lower dose of gene therapymight be effective. However, these experiments were limited by theoccurrence of infection and the small number of groups. The nextexperiment determined whether a single high dose of ADV-VEGF would beeffective in larger wounds.

[0081] This experiment tested the hypothesis that ADV-VEGF will beeffective in accelerating wound closure, even when the size of the woundis increased, with metabolic control ascertained (therefore female micewere utilized). Since patients usually present with larger wounds, thesize of the wound was doubled to 1.4 cm which represents 35% of thetotal diameter of the mouse dorsum. Four treatment groups at 0.5 logdifferences and two control groups were tested. One set of controls wasinjected with saline at the time of injection and the other withADV-DL312 5×10¹⁰ particles/wound.

[0082] The only group showing a statistically significant accelerationof closure was the group treated with 5×10¹¹ VPs ADV-VEGF (which was themaximum dose tested). Therefore, it is necessary to determine if agreater acceleration of healing can be obtained. Another limitation ofthis experiment was that these mice were 12-14 weeks old, older miceexhibit poor metabolic control. The glucose levels in all groups werebetween 300-310. This is too high and may have affected the results,since this places the mice in a particularly catabolic state, which isnot analogous to the clinical situation.

EXAMPLE 7 Determination of Toxicity (10 Mice per Group)

[0083] A comprehensive examination was initiated to look for indicatorsof toxicity. The liver was examined for any signs of hepatitis. Thelungs were examined because angiogenic therapy can potentially causepetechia in lungs and bleeding. The kidneys were examined at thehistological level because nephropathy is very common in diabetics andcould be precipitated by angiogenic therapy. The cecum was analyzedbecause it is the most rapidly proliferating organ in the body and anytherapy that may effect a wound could also effect the rapidly dividingepithelium. The retina was examined closely because it is of greatconcern whether systemic therapy could cause retinopathy. In addition,the heart was analyzed because of concerns that angiogenic therapy couldadversely effect this muscle. The brain was examined because it is knownthat angiogenic therapy can cause petechia and bleeds in the brain,which is particularly susceptible in the diabetic CNS vasculature.

[0084] There were no pathologic findings in any organs 90 days after5×10¹¹ VPs of ADV-VEGF was administered into the diabetic ulcer. In theliver, there was neither acute hepatitis from the ADV nor proliferationof the sinusoidal lining of the endothelial cells from the VEGF. Thekidneys, lungs, retina, or cecum had neither hemorrhage nor any otherpathologic findings. Similarly, neither the heart or brain (not shown)had any abnormal pathologic findings. This is significant, because inpast experience, angiogenic therapy was reported to have a significanteffect on the immune system, however no gross difference in weight,size, or histological examination of the organs were manifested.

[0085] At this point, the experiment was limited, because only 3 timepoints were assayed and, at most, only 5 mice were tested at any timepoint. The consistency of the results suggests that they can beduplicated in significantly higher numbers. After 3 months a toxiceffect may have been realized, but it was transient.

[0086] It was then determined whether VEGF levels in the serum ofdiabetic mice were increased after ADV-VEGF local administration. VEGFserum levels were determined by a commercially available ELISA kit inaccordance with the manufacturer's protocol. It is emphasized that humanVEGF was assayed in the serum. A linear VEGF standard curve wasgenerated for each assay (r=0.99 to 1 between calculated and measuredVEGF levels) with the supplied recombinant VEGF protein in the rangefrom 10000 pg/ml to 15.6 pb/ml and serum levels were calculatedaccordingly. Only 3-5 mice per group were studied, enough to determinethat the assay was accurate and consistent. VEGF levels demonstrated apeak of activity in day 10 after the wound was made. However, there wereno differences in the control group treated with saline versus theADV-VEGF group.

[0087] Utilizing the same series of mice, larger quantifies were studied(5×10¹¹ vp of ADV-VEGF and 5×10¹¹ vp of ADV-DL312 vs. PBS, respectively)and the effects on the organ weights of the lungs, kidneys, and spleenmeasured. No differences were found for tested time points. However, thesampling size is too small to draw any definitive conclusions.Preliminary data from days 5, 10, 15 and 90 5×10¹¹ ADV-VEGF, 5×10¹¹ADV-DL³¹² vs. PBS, respectively) of serum chemistries: ALT, AST, ALP,Bilirubin, Creatinine, Glucose, BUN, Platelets, WBC, and Hemoglobin showno differences detected to date.

[0088] In summary, the preliminary data suggest that 5×10¹¹ vp ofADV-VEGF accelerates ulcer healing in C57BL/KsJ-db/db mice. VEGFstimulates angiogenesis and accelerates the healing process as evidencedby:

[0089] 1) a reduced time to closure (a healed wound is defined as 100%epithelialization and no drainage) in experimental diabetic ulcers, and

[0090] 2) accelerated epithelialization and increased cellular density.

[0091] The preliminary data suggest that a high dose of 5×10¹¹ ADV-DL312VPs/wound is effective. However, such a high dose of ADV particlesunlikely to be utilized in patients in years to come because of concernsof potential toxicity.

[0092] Modifications and variations of the present invention will beobvious to those skilled in the art from the foregoing detaileddescription. Such modifications and variations are intended to comewithin the scope of the following claims.

1 2 1 36 DNA Homo sapiens 1 cccaagcttg ccgccgccat gaactttctg ctgtct 36 229 DNA Homo sapiens 2 gctctagaat ctggttcccg aaaccctga 29

I claim:
 1. A method for tissue engineering a diabetic ulcer comprisingadministering to the wound an effective amount of VEGF for a sustainedperiod of time of at least two weeks to enhance the rate of closure ofthe wound.
 2. The method of claim 1 wherein the VEGF is administered inthe form of a nucleic acid molecule encoding the VEGF.
 3. The method ofclaim 1 wherein the VEGF reverses bacterial contamination of the woundin a patient with a diabetic ulcer.
 4. The method of claim 2 wherein thenucleic acid molecule is a viral vector.
 5. The method of claim 4further comprising administering the adenoviral vector at aconcentration of at least 1.6×10¹⁰ adenoviral-VEGF particles.
 6. Themethod of claim 4 further comprising administering the adenoviral vectorat a concentration of at least 5×10¹⁰ adenoviral-VEGF particles.
 7. Themethod of claim 4 further comprising administering the adenoviral vectorat a concentration of at least 5×10¹¹ adenoviral-VEGF particles.
 8. Themethod of claim 2 wherein the nucleic acid molecule is in the form ofgenetically engineered cells secreting VEGF.
 9. The method of claim 2wherein the nucleic acid molecule is in the form of naked DNA encodingVEGF.
 10. The method of claim 1 wherein the VEGF is administered by asustained delivery pump.
 11. The method of claim 1 wherein the VEGF isadministered in a sustained delivery polymeric device.
 12. The method ofclaim 8 wherein the device is formed of a biodegradable polymer in theform of microspheres, slabs, disks, or gels.
 13. The method of claim 1wherein the VEGF is released over a period of between two and six weeks.14. A composition for tissue engineering a diabetic ulcer comprising aneffective amount of VEGF administered for a sustained period of time ofat least two weeks to enhance the rate of closure of the wound.
 15. Thecomposition of claim 14 wherein the VEGF is in the form of a nucleicacid molecule encoding the VEGF.
 16. The composition of claim 14 whereinthe VEGF is in the form of a viral vector encoding the VEGF.
 17. Thecomposition of claim 15 wherein the nucleic acid molecule is in the formof genetically engineered cells secreting VEGF.
 18. The composition ofclaim 15 wherein the nucleic acid molecule is in the form of naked DNAencoding VEGF.
 19. The composition of claim 14 wherein the VEGF isformulated in a sustained delivery pump.
 20. The composition of claim 14wherein the VEGF is formulated in a sustained delivery polymeric device.21. The composition of claim 20 wherein the device is formed of abiodegradable polymer in the form of microspheres, slabs, disks, orgels.